Enhanced CHOLESTEROL biosynthesis promotes breast cancer metastasis via modulating CCDC25 expression and neutrophil extracellular traps formation

Neutrophil extracellular traps (NETs) has been demonstrated to regulate the metastasis of breast cancer. In this study, we showed that de novo cholesterol biosynthesis induced by ASPP2 depletion in mouse breast cancer cell 4T1 and human breast cancer cell MDA-MB-231 promoted NETs formation in vitro, as well as in lung metastases in mice intravenously injected with ASPP2-deficient 4T1 cells. Simvastatin and berberine (BBR), cholesterol synthesis inhibitors, efficiently blocked ASPP2-depletion induced NETs formation. Cholesterol biosynthesis greatly enhanced Coiled-coil domain containing protein 25 (CCDC25) expression on cancer cells as well as in lung metastases. CCDC25 expression was co-localized with caveolin-1, a lipid raft molecule, and was damped by inhibitor of lipid rafts formation. Our data suggest that cholesterol biosynthesis promotes CCDC25 expression in a lipid raft-dependent manner. Clinically, the expression of CCDC25 was positively correlated with the expression of 3-hydroxy-3-methylglutaryl-CoAreductase (HMRCG), and citrullinated histone H3 (H3cit), in tissues from breast cancer patients. High expression of CCDC25 and HMGCR was related with worse prognosis in breast cancer patients. In conclusion, our study explores a novel mechanism for de novo cholesterol biosynthesis in the regulation of CCDC25 expression, NETs formation and breast cancer metastasis. Targeting cholesterol biosynthesis may be promising therapeutic strategies to treat breast cancer metastasis.

Real-time PCR was completed using SYBR Green PCR Master Mix (Takara, Otsu, Japan) on QuantStudioTM 7 Flex (Thermo Fisher Scientific). All target genes were normalized to cyclophilin B. Gene expression was quantified using the delta Ct method.
Primer sequences were as follows: medium was added to each well after washing with PBS, and then 2 μg/ml polybrene has been adjuncted before 10, 15, and 20l of virus were added respectively. After 72 hours of culture, cellular RNA was extracted for RT-PCR detection to analyze infection efficiency and optimal MOI value. All lentiviruses were designed with three siRNA sequences and synthesized three shRNAs. The most suitable sequences were verified and selected for subsequent experiments. In the subsequent culture and experiments of lentivirus-infected cells, 2μg/ml puromycin was added from time to time to construct a stable lentivirus strain.

Isolation of neutrophils
Mouse neutrophils were collected from female BALB/c mice. Neutrophil isolation kit (2013NM, TBD, Tianjin, China) was used to extract and isolate neutrophils from mouse femur and tibia. Use homogenate to rinse out the lumen. After blowing and beating into a single cell suspension, filter with a 70um filter. Centrifuge at 500g for 10min, resuspend the cells with erythrocyte sedimentation solution, and add it to the separation solution of the concentration gradient. 800g, centrifuge for 30min. Take the middle cell layer and add 3 times its volume of washing solution to resuspend. Centrifuge at 400g for min. Add erythrocyte lysis solution to remove red blood cells. After centrifugation, add washing solution to wash and centrifuge again to obtain neutrophil pellet. Add appropriate amount of DMEM medium to resuspend, and follow-up experiments can be carried out.

Wound-Healing assay
Tumor cells were culture in 6-well plant, until cell density reached to 90%. 10μl pipette tip was used to scar the cells. Cell wound healing ability was calculated using a Leica microscope at 0h, 12h and 24h. All assays were conducted three times.

Immunohistochemical staining
Briefly, the slide is dewaxed, hydrated, quenched endogenous peroxidase activity, Subsequently, the slides were counterstained with hematoxylin and dehydrated in graded alcohol and mounted. The expression of CCDC25, Caveolin-1, HMGCR, MPO and H3cit was scored according to the signal intensity and distribution. We used a two-level scoring method, staining degree 0-3 points (negative, light yellow, light brown, tan), and positive range 1-4 points (0-25%, 26%-50%, 51%-75%, 76%-100 %). The staining degree points were multiplied with positive range points to produce a final score for each case. Tissues with immunohistochemical scoring 2 or less were considered as low, 3 to 12 as high. The immunostaining evaluation was performed independently by two experienced pathologists.

Measurement of serum MPO-DNA level
Anti-MPO monoclonal antibody (5μg/ml, R&D) was coated on a 96-well plate overnight at 4℃. Blocked with 1% BSA for 1h at room temperature then added 100l serum diluted at 1:5 with normal saline, incubate at room temperature for 2h. After washed with PBST for five times, PicoGreen® dsDNA Quantitation Reagent (YEASEN, Shanghai, China) was used to measure MPO-DNA content based on the manufacturer's protocol. Data were normalized to healthy mouse serum for each test.
All assays were conducted at least 3 times.

Supplementary Ta ble1
The clinicopathologic characteristics of 60 breast cancer patients